Prey range characterization, ribotyping, and diversity of soil and rhizosphere Bdellovibrio spp. isolated on phytopathogenic bacteria

Abstract

Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil. Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios. Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp. carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A. tumefaciens was used as prey. In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 x 10(2) to 6 x 10(3) and 2.8 x 10(2) to 2.3 x 10(4) PFU g(-1). A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells. An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination. Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively. The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced. One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups.

FIG. 1

Kinetics of lysis of P. corrugata by strains BEP2 and BRP4. The control contained prey cells without bdellovibrios. Bars indicate standard errors.

FIG. 2

Electron micrographs of Bdellovibrio isolates. (a) BEP2 attack-phase cell. (b) TRA2 attack-phase cells (TR), near an A. tumefaciens substrate cell (At). (c) BRP4 attack-phase cell. (d) SRP1 attack-phase cells (SR). Pc, P. corrugata prey cell; Bp, bdelloplast. Bars = 0.5 μm.

FIG. 3

Restriction digestion of PCR-amplified 16S rDNA fragments of soil and rhizosphere Bdellovibrio strains obtained by using Bacteria-specific primer 63F and Bdellovibrio-specific primer 842R. (a) Soil strains SRP1, SRP2, SRP3, SRP4, and UKi2. Lanes 1 through 5, BamHI; lanes 6 through 10, EcoRI; lanes 11 through 15, SacII; lanes 16 through 20, XbaI. (b) Soil strains SRE6, SRE7, SRE8, SRE9, and SRE12. Lanes 1 through 5, BamHI; lanes 6 through 10, EcoRI; lanes 11 through 15, SacII; lanes 16 through 20, XbaI. (c) Rhizosphere strains BEP2, BRP4, TRA2, and soil strain 109J. Lanes 1 through 4, BamHI; lanes 5 through 8, EcoRI; lanes 9 through 12, SacII; lanes 13 through 16, XbaI. (d) Rhizosphere strains BEP1, BEP3, BEP4, BRP3, and W. Lanes 1 through 5, BamHI; lanes 6 through 10, EcoRI; lanes 11 through 15, SacII; lanes 16 through 20, XbaI. Lanes MW contained the molecular weight marker.

FIG. 4

16S rRNA-based phylogenetic tree for Bdellovibrio isolates and previously published sequences. The tree was produced by using the neighbor-joining algorithm and sequence positions at which the residues in at least 50% of all available complete or almost complete 16S rRNA sequences from members of this subgroup were identical. The orders of branching were similar for all of the treeing approaches used (see Materials and Methods). Bar = estimated 10% sequence divergence. The bootstrap values at the nodes are percentages based on 100 iterations; only values greater than 50% are shown.

FIG. 5

Southern analysis of the hit locus following SauIIIA restriction of total DNA and hybridization with a digoxigenin-labelled hit probe obtained from B. bacteriovorus 109J. Lane 1, molecular weight markers; lane 2, strain 109J; lane 3, strain BEP2; lane 4, strain BRP4; lane 5, strain SRP1; lane 6, strain TRA2; lane 7, homologous probe.